Therapeutic agents for alzheimer&#39;s disease and cancer

ABSTRACT

To provide a therapeutic drug for Alzheimer&#39;s disease and/or a cancer. 
     The therapeutic drug for Alzheimer&#39;s disease and/or a cancer contains an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative.

TECHNICAL FIELD

The present invention relates to a therapeutic drug for Alzheimer's disease and/or a cancer, the drug containing an anti-nicastrin antibody.

BACKGROUND ART

In Japan, the three most common death-causing diseases are cancer (30.3%), cardiac disease (15.3%), and cerebrovascular disease (15.2%). As the population ages, the percentage of patients with such diseases increases, which greatly affects medical costs required for treatment or nursing care. In recent years, a nursing-care insurance system for cerebrovascular disease patients has been established as a national policy.

In Japan, the number of deaths from cancer was 320,315 (i.e., 253.9 per 100,000) in 2004. In Japan, in 2003, lung cancer (22.3%) was ranked first among cancer deaths in men, followed by gastric cancer (17.2%) and liver cancer (12.5%), whereas colon cancer (14.6%) was ranked first among cancer deaths in women, followed by gastric cancer (14.2%) and lung cancer (12.3%). According to a report by the National Cancer Center in Japan, regarding five-year survival rates for major types of cancer, the five-year survival rate of pancreatic cancer patients is the lowest (only a few percent), followed by that of patients with gallbladder cancer, lung and bronchial cancer, liver cancer, esophageal cancer, etc. Ohno, Nakamura, et al., have estimated that the number of new cases of male cancers will be 501,000 in 2020 (major sites of cancer: lung, prostate gland, stomach, colon, liver, etc.), whereas the number of new cases of female cancers will be 337,000 in 2020 (major sites of cancer: breast, colon, stomach, lung, uterus, rectum, liver, etc.). Thus, cancer is predicted to become a major death-causing disease in future (as is the case at present), and development of a therapy for cancer is essential.

Cancer therapy has changed with the times. Recently, in addition to hitherto performed surgery, drug therapy, and radiotherapy, endoscopic resection of cancer tissue has been carried out, and chemotherapy for outpatients has been performed more and more. However, about 40% of cancer cases are treated through surgery at present, and radiotherapy or chemotherapy is less effective for some cancers (e.g., pancreatic cancer). In some cancer cases, chemotherapy can reduce a size of cancer tissue, but encounters difficulty in completely curing the disease. In many refractory cancer cases, adverse reactions to an anticancer agent (i.e., side effects thereof) are more pronounced than the effects of the drug.

Cerebrovascular diseases are classified into a cognitive disorder, which is caused by vascular disorder, and Alzheimer's disease, which is a neurodegenerative disease. In Japan, a number of patients with dementia caused by Alzheimer's disease (AD) has increased with adoption of Europeanized and Americanized meals and aging of the population.

AD is a neurodegenerative disease which develops various intellectual dysfunctions (including memory impairment) due to degeneration or loss of cerebral cortical neurons. An AD brain is characterized by accumulation of an abnormal protein called “β-amyloid,” which is closely related to loss of neurons (Non-Patent Document 1).

β-Amyloid is accumulated in an AD brain in the pathological form of senile plaque or vascular amyloid. From the biochemical viewpoint, β-amyloid is formed of Aβ peptide including 40 to 42 amino acid residues. Aβ is produced from APP (amyloid precursor protein) through two-step cleavage and is secreted extracellularly. In the second step, a C-terminal fragment of APP is cleaved at an intramembrane site by the protease activity of the enzyme γ-secretase, and the thus-formed Aβ is released extracellularly. Cleavage of the C-terminal fragment of APP occurs at different sites; i.e., at position 40 (90%) and at position 42 (10%) (Non-Patent Document 2). Aβ42 is more highly aggregated in the form of β-amyloid and is preferentially accumulated in an AD brain from an early stage (Non-Patent Document 3).

As has been shown, presenilin (PS) protein, which is an expression product of a major pathogenic gene of familial AD, corresponds to a catalytic subunit of γ-secretase, which is a membrane-associated aspartic protease (Non-Patent Documents 4 and 5).

γ-Secretase has been shown to be involved not only in AD but also in Notch signaling (Non-Patent Document 6). As has been known, a γ-secretase inhibitor (i.e., a low-molecular-weight compound) induces apoptosis in Kaposi's sarcoma (Non-Patent Document 7) or inhibits survival of T-ALL cells (Non-Patent Document 8). However, it has been reported that a γ-secretase inhibitor may promote malignant transformation (Non-Patent Document 9). Thus, inhibition of Notch signaling does not necessarily induce cell death in all cancers, and in the future studies will be carried out to determine whether or not a γ-secretase inhibitor can be used as a therapeutic drug for cancer.

Under such circumstances, γ-secretase has been considered important as a therapeutic target for AD or cancer, but a cancer therapeutic drug based on γ-secretase has not successfully been developed for, for example, the following reason. Since γ-secretase is a complex formed of a plurality of membrane proteins and exhibits protease activity in the membrane, difficulty is encountered in reconstituting γ-secretase while maintaining protease activity, and drug screening is not properly carried out by use of γ-secretase.

As has been known, human active γ-secretase complex is a large membrane protein complex having a molecular weight of 250 to 500 kDa or more and including the following four proteins: presenilin, nicastrin (NCT), APH-1, and PEN-2. That is, nicastrin is a constituent molecule of γ-secretase. Many attempts have been made to search for γ-secretase activity inhibitors by use of low-molecular-weight compounds, but no report has been provided to show a result of an experiment by use of an anti-nicastrin antibody for development of a γ-secretase activity inhibitor or a therapy for AD and/or cancer. Although there are many AD and cancer patients, a good drug for a treatment of the diseases has not yet been provided. Development of a therapeutic drug for AD or cancer could reduce burden of nursing care as a matter of course, along with medical costs.

-   Non-Patent Document 1: Selkoe D J., Physiol. Rev. 2001, 81 (2):     741-766, Alzheimer's disease: genes, proteins, and therapy -   Non-Patent Document 2: Suzuki N., et al. Science 264: 1336, 1994 -   Non-Patent Document 3: Iwatsubo T., Odaka A., Suzuki N., Mizusawa     H., Nukina N., Ihara Y., Neuron. 1994, 13 (1): 45-53 -   Non-Patent Document 4: Wolfe M S., Xia W., Ostaszewski B L., Diehl T     S., Kimberly W T., Selkoe D J. (1999), Nature 398 (6727): 513-517 -   Non-Patent Document 5: Li Y M., Xu M., Lai M T., Huang Q., Castro J     L., DiMuzio Mower J., Harrison T., Lellis C., Nadin A., Neduvelil J     G., Register R B., Sardana M K., Shearman M S., Smith A L., Shi X     P., Yin K C., Shafer J A., Gardell S J. (2000), Nature 2000 Jun. 8,     405 (6787): 689-94 -   Non-Patent Document 6: J. Biol. Chem. 2001 Aug. 10; 276 (32):     30018-30023 -   Non-Patent Document 7: Oncogene. 2005 Sep. 22; 24 (42): 6333-6344 -   Non-Patent Document 8: Mol. Cell. Biol. 2003 January; 23 (2):     655-664 -   Non-Patent Document 9: Br. J. Cancer. 2005 Sep. 19; 93 (6): 709-718

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention provides a new therapeutic drug for AD or a cancer. The present invention also provides a screening method for selecting such a therapeutic drug.

Means for Solving the Problems

The present inventors have succeeded in expressing active γ-secretase by use of budding baculovirus (see WO 2005/038023). The present inventors have screened anti-nicastrin antibodies on a basis of active γ-secretase activity by use of budding baculovirus, and as a result have found that an excellent anti-nicastrin antibody is useful as a therapeutic drug for AD and/or a cancer, since the anti-nicastrin antibody exhibits γ-secretase-neutralizing activity and inhibits proliferation of Notch-expressing cells and/or improves survival rate. The present invention has been accomplished on the basis of this finding. Also, the present inventors have found that an anti-nicastrin antibody inhibits reaction between nicastrin and a γ-secretase substrate (i.e., a polypeptide including the intramembrane sequence of a receptor or APP), and thus this reaction system can be employed for selecting, through screening, an antibody which inhibits γ-secretase activity. The present invention has been accomplished also on the basis of this finding.

Accordingly, the present invention provides a therapeutic drug for AD and/or a cancer containing an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative.

The present invention also provides a screening method for selecting an antibody which inhibits γ-secretase activity, characterized by comprising reacting nicastrin with a γ-secretase substrate in a presence of a test antibody.

The present invention also provides use of an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative for producing a therapeutic drug for Alzheimer's disease and/or a cancer.

The present invention also provides a method for treatment of Alzheimer's disease and/or a cancer, characterized by comprising administering an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative to a subject in need thereof.

Effects of the Invention

According to the therapeutic drug for AD and/or a cancer of the present invention, γ-secretase activity can be inhibited by an anti-nicastrin antibody, to thereby treat Alzheimer's disease and/or a cancer.

According to the screening method of the present invention, the reaction system between nicastrin and a γ-secretase substrate can be employed for selecting an antibody which inhibits γ-secretase activity; i.e., an antibody effective for the treatment of Alzheimer's disease and/or a cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of screening of anti-nicastrin antibodies through BV-ELISA.

FIG. 2 shows results of screening of anti-nicastrin antibodies through western blot analysis by use of BV.

FIG. 3 shows results of western blot analysis of various forms of nicastrin expressed in COS-7 cells.

FIG. 4 shows results of an experiment for evaluating cross-reactivity of anti-nicastrin antibodies to deglycosylated nicastrin (“O” represents Endo H-resistant nicastrin; “black dot” represents completely deglycosylated nicastrin; and “Δ” represents neuraminidase-desialylated nicastrin).

FIG. 5 shows results of IP of nicastrin from a soluble membrane fraction of HeLa cell by use of anti-nicastrin antibodies.

FIG. 6 shows results of treatment of a HeLa cell lysate with trypsin.

FIG. 7 shows results of immunostaining of HeLa cells by use of anti-nicastrin antibodies.

FIG. 8 shows results of immunostaining of NKO cells and NKO/NCT cells by use of anti-nicastrin antibodies.

FIG. 9 shows results of co-staining by use of anti-nicastrin antibodies and antibodies to various marker proteins.

FIG. 10 shows results of co-staining by use of anti-nicastrin antibodies and cholera toxin subunit B (CTB).

FIG. 11 shows an effect of anti-nicastrin antibodies on in vitro γ-secretase activity.

FIG. 12 shows an effect of DAPT on a viability of HeLa cells or A549 cells.

FIG. 13 shows an effect of inhibition of nicastrin expression on a viability of A549 cells.

FIG. 14 shows results of inhibition of expression of endogenous nicastrin in A549 cells by siRNA.

FIG. 15 shows an effect of anti-nicastrin antibodies on the viability of A549 cells.

FIG. 16 shows mutation sites of Notch1 gene in various T-ALL-derived cells.

FIG. 17 shows results of western blot analysis of Notch1 gene products in various T-ALL-derived cell lysates.

FIG. 18 shows an effect of a γ-secretase inhibitor YO on proliferation of various T-ALL-derived cells.

FIG. 19 shows an effect of an anti-nicastrin antibody on proliferation of DND-41 cells.

FIG. 20 shows results of western blot analysis of anti-nicastrin antibodies and a nicastrin-N100 fraction.

FIG. 21 shows an effect of anti-nicastrin antibodies on inhibition of binding between nicastrin and N100-FLAG.

FIG. 22A shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.

FIG. 22B shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.

FIG. 22C shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will next be described in detail.

The present invention is directed to a therapeutic drug for AD and/or a cancer containing an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative; to use of the antibody, the derivative, or the fragment for producing such a therapeutic drug; and to a method for the treatment of Alzheimer's disease and/or a cancer.

In the present invention, as described hereinbelow, the anti-nicastrin antibody derivative encompasses a modified anti-nicastrin antibody and an anti-nicastrin antibody to which a compound exhibiting a desired pharmaceutical activity has been bound.

Nicastrin is a membrane protein and forms a complex to exhibit γ-secretase activity. An amino acid sequence of nicastrin and a sequence of the gene coding therefor are disclosed in GenBank number (NM_(—)015331) (SEQ ID Nos: 1 and 2). As used herein, “nicastrin protein” encompasses both full-length nicastrin protein and a fragment of nicastrin. As used herein, “fragment of nicastrin” refers to a polypeptide which includes a predetermined region of nicastrin protein and which does not necessarily have a function of natural nicastrin protein.

Nicastrin protein, which is employed as an antigen in the present invention, is preferably human nicastrin protein, but is not necessarily limited thereto. The nicastrin protein employed in the present invention may be nicastrin derived from any non-human species, such as canine nicastrin, feline nicastrin, mouse nicastrin, hamster nicastrin, or drosophila nicastrin. Preferably, an antibody selected by use of nicastrin protein neutralizes human active γ-secretase including nicastrin as a constituent molecule.

Human active γ-secretase is a large-molecule membrane protein complex having a molecular weight of 250 to 500 kDa or more and including the following four proteins: fragmented presenilin, nicastrin, APH-1, and PEN-2.

The active γ-secretase employed in the present invention may be prepared through any of methods described in the Examples hereinbelow (WO 2005/038023). Natural human active γ-secretase may be prepared from a human brain homogenate, but is very difficult to employ for screening of γ-secretase inhibitors. Therefore, the active γ-secretase employed is preferably prepared through the method by the present inventors for successfully expressing an active γ-secretase complex by use of budding baculovirus (see WO 2005/038023).

In the present invention, γ-secretase activity is determined through a method by Yasuko Takahashi, et al. (J. Biol. Chem. 2003 May 16; 278 (20): 18664-70), which is a generally known method. Specifically, a test substance is mixed with microsomes (serving as an enzyme) prepared from brain tissue or cultured cells, and the mixture is incubated at 4° C. for 12 hours. Subsequently, 1 μM C100FmH serving as a substrate is added to the reaction mixture, and the mixture is incubated at 37° C. for 12 hours. Thereafter, an amount of amyloid-β is measured through sandwich ELISA, to thereby determine γ-secretase activity.

Preparation of Anti-Nicastrin Antibody

Preferably, the anti-nicastrin antibody employed in the present invention not only binds specifically to nicastrin protein, but also neutralizes human active γ-secretase. No particular limitation is imposed on an origin, type (monoclonal or polyclonal), and form of the anti-nicastrin antibody. Specifically, the anti-nicastrin antibody may be a known antibody such as a mouse antibody, a rat antibody, an avian antibody, a human antibody, a chimera antibody, and a humanized (CDR-grafted) antibody. The anti-nicastrin antibody is preferably a human, chimera, or humanized monoclonal antibody.

Examples of the anti-nicastrin monoclonal antibody include a monoclonal antibody produced by a hybridoma, and a monoclonal antibody produced in a host transformed with an expression vector containing a gene for the antibody through a genetic engineering technique.

Basically, a hybridoma which produces the monoclonal antibody may be prepared through a known technique as described below. Specifically, the hybridoma may be prepared through the following procedure: a mammal is immunized with nicastrin protein serving as a sensitizing antigen through a customary immunization method; the resultant immunocyte is fused with a known parental cell through a customary cell fusion method; and a cell for producing the monoclonal antibody is selected through a customary screening method.

Specifically, the monoclonal antibody can be prepared as follows.

Firstly, nicastrin protein, which is employed as a sensitizing antigen for preparing the monoclonal antibody, is obtained through expression of a nicastrin gene/amino acid sequence disclosed in GenBank number (NM_(—)015331). Specifically, an appropriate host cell is transformed with a known expression vector system containing the gene sequence encoding nicastrin, and then human nicastrin protein of interest is purified from the resultant host cell or a culture supernatant of the cell through a known method. Alternatively, natural nicastrin protein may be employed after being purified.

Subsequently, the thus-purified nicastrin protein is employed as a sensitizing antigen. Alternatively, a partial peptide of the nicastrin protein may be employed as a sensitizing antigen. Such a partial peptide may be obtained through chemical synthesis on the basis of the amino acid sequence of nicastrin protein, through integration of a portion of the nicastrin gene into an expression vector, or through degradation of natural nicastrin protein by use of protease. No particular limitation is imposed on a site or size of a nicastrin protein portion employed as a partial peptide.

No particular limitation is imposed on the mammal which is immunized with the sensitizing antigen, but preferably, the mammal is selected in consideration of compatibility of the resultant immunocyte with a parental cell employed for cell fusion. In general, a rodent (e.g., mouse, rat, or hamster), avian, rabbit, monkey, or the like is employed.

Immunization of an animal with the sensitizing antigen is carried out through a known method. For example, in a generally employed immunization method, the sensitizing antigen is intraperitoneally or subcutaneously injected into a mammal. Specifically, the sensitizing antigen is diluted by PBS (phosphate-buffered saline), saline, or the like, to form a suspension of an appropriate volume. If desired, the resultant suspension is mixed with an appropriate volume of a common adjuvant (e.g., Freund's complete adjuvant). After emulsification of the resultant mixture, the emulsion is administered to a mammal several times every 4 to 21 days. Upon immunization with the sensitizing antigen, an appropriate carrier may be employed. Particularly when the sensitizing antigen is a partial peptide of low molecular weight, preferably, the partial peptide employed for immunization is bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

After immunization of a mammal as described above, and following confirmation of an increase in serum level of an antibody of interest, immunocytes are collected from the mammal and then subjected to cell fusion. The type of immunocytes is particularly preferably splenocyte.

A mammalian myeloma cell is employed as a parental cell which is fused with the aforementioned immunocyte. The myeloma cell employed is preferably a known cell line; for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D. H., et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M., et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F., et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), or R210 (Galfre, G., et al., Nature (1979) 277, 131-133).

Cell fusion between the aforementioned immunocyte and myeloma cell may be basically carried out through a known method, such as a method of Kohler, Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

More specifically, the aforementioned cell fusion is carried out in a common nutrient culture medium in the presence of, for example, a cell fusion promoter. Examples of the cell fusion promoter employed include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an auxiliary agent (e.g., dimethyl sulfoxide) may be further added in order to enhance cell fusion efficiency.

The ratio of the immunocyte and myeloma cell employed may be determined as desired. For example, an amount of the immunocyte is preferably 1 to 10 times that of the myeloma cell. Examples of the culture medium which may be employed for the aforementioned cell fusion include RPMI 1640 medium and MEM medium, which are suitable for proliferation of the aforementioned myeloma cell line; and culture media which are generally employed for such a cell culture. Such a culture medium may be employed in combination with a serum supplement such as fetal calf serum (FCS).

In the cell fusion, predetermined amounts of the aforementioned immunocyte and myeloma cell are well-mixed in any of the aforementioned culture media, and a solution of PEG (e.g., PEG having an average molecular weight of about 1,000 to about 6,000) which has been heated in advance to about 37° C. is added to the resultant mixture in a predetermined amount (generally 30 to 60% (w/v)), followed by mixing, to thereby yield a hybridoma of interest. Subsequently, a procedure including sequential addition of an appropriate culture medium and removal of a supernatant obtained through centrifugation is repeated, to thereby remove substances (e.g., a cell fusion promoter) which are not suitable for growth of the hybridoma.

Separation of the thus-yielded hybridoma is carried out through culturing in a common selective culture medium such as a HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). A culturing in the aforementioned HAT medium is continued for a sufficient period of time (generally several days to several weeks) for apoptosis of cells (i.e., non-fused cells) other than the hybridoma of interest. Subsequently, a customary limiting dilution technique is performed for screening and monocloning of the hybridoma which produces a target antibody.

An antibody which recognizes nicastrin protein may be prepared through a method described in WO 03/104453.

Screening and monocloning of a target antibody may be carried out through a known screening method on a basis of antigen-antibody reaction. For example, an antigen is bound to a carrier (e.g., beads made of polystyrene or a similar material, or a commercially available 96-well microtiter plate) and then reacted with a culture supernatant of the hybridoma, and subsequently the carrier is washed, followed by reaction with, for example, an enzyme-labeled secondary antibody, to thereby determine whether or not the culture supernatant contains a target antibody which reacts with a sensitizing antigen. Cloning of the hybridoma which produces a target antibody may be performed through, for example, a limiting dilution technique. In this case, the antigen may be an antigen employed in immunization.

In addition to preparation of the aforementioned hybridoma through immunization of a non-human animal with an antigen, a human antibody of interest having binding activity to nicastrin may be prepared by sensitizing human lymphocyte with nicastrin in vitro, and fusing the thus-sensitized lymphocyte with a human-derived myeloma cell having permanent division capacity (see JP-A-01-059878). Alternatively, nicastrin serving as an antigen may be administered to a transgenic animal having all of the human antibody gene repertories, to thereby yield a cell which produces an anti-nicastrin antibody, and a human antibody against nicastrin may be obtained from the cell after it has been immortalized (see WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).

The thus-prepared monoclonal-antibody-producing hybridoma can be subcultured in a common culture medium and can be stored in liquid nitrogen for a long period of time.

A monoclonal antibody is produced from the hybridoma through, for example, a method in which the hybridoma is cultured by a customary technique, and the monoclonal antibody is obtained from the resultant culture supernatant; or a method in which the hybridoma is administered to a mammal exhibiting compatibility with the hybridoma to thereby proliferate the hybridoma, and the monoclonal antibody is obtained from ascitic fluid of the mammal. The former method is suitable for obtaining a monoclonal antibody of high purity, whereas the latter method is suitable for a mass production of a monoclonal antibody.

The monoclonal antibody employed in the present invention may be a recombinant antibody. Such a recombinant antibody is produced through the following procedure: the antibody gene is cloned from the hybridoma; the gene is integrated into an appropriate vector; and the vector is introduced into a host, followed by production of the recombinant antibody through a genetic recombination technique (see, for example, Vandamme, A. M., et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).

Specifically, mRNA encoding a variable (V) region of an anti-nicastrin antibody is isolated from the hybridoma which produces the anti-nicastrin antibody. Isolation of mRNA is carried out as follows. Total RNA is prepared through a known method such as the guanidine ultracentrifugation method (Chirgwin, J. M., et al., Biochemistry (1979) 18, 5294-5299) or the AGPC method (Chomczynski, P., et al., Anal. Biochem. (1987) 162, 156-159), and target mRNA is prepared by means of, for example, mRNA Purification Kit (product of Pharmacia). Alternatively, mRNA may be directly prepared by means of QuickPrep mRNA Purification Kit (product of Pharmacia).

The thus-obtained mRNA is employed for synthesis of cDNA of the antibody V region by use of reverse transcriptase. Synthesis of cDNA is carried out by means of, for example, AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (product of Seikagaku Corporation). Alternatively, synthesis and amplification of cDNA may be carried out by means of, for example, 5′-Ampli FINDER RACE Kit (product of Clontech) or the 5′-RACE method using PCR (Frohman, M. A., et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A., et al., Nucleic Acids Res. (1989) 17, 2919-2932).

A target DNA fragment is purified from the resultant PCR product and ligated to vector DNA. Subsequently, a recombinant vector is prepared from the vector DNA and then introduced into Escherichia coli or the like, followed by colony selection, to thereby prepare a recombinant vector of interest. The nucleotide sequence of the target DNA fragment is determined through a known method such as a dideoxynucleotide chain termination method.

DNA encoding the V regions of a target anti-nicastrin antibody is obtained, and then the DNA is integrated into an expression vector containing DNA encoding constant regions (C regions) of the target antibody.

In order to produce the anti-nicastrin antibody employed in the present invention, the gene for the antibody is integrated into an expression vector so that the gene can be expressed under a control of an expression regulatory region (e.g., an enhancer or a promoter). Subsequently, a host cell is transformed with this expression vector for expression of the antibody.

The gene for the antibody may be expressed by transforming a host cell with both an expression vector containing the DNA encoding a heavy chain (H chain) of the antibody and an expression vector containing the DNA encoding a light chain (L chain) of the antibody, or by transforming a host cell with a single expression vector containing the DNA encoding the heavy and light chains of the antibody (see WO 94/11523).

In addition to the aforementioned host cell, a transgenic animal may be employed for production of a recombinant antibody. For example, an antibody gene is inserted into a gene encoding a protein produced specifically in milk (such as goat β-casein) to prepare a fusion gene. A DNA fragment including the fusion gene having the inserted antibody gene is injected into an embryo of a goat, and this embryo is implanted into a female goat. An antibody of interest is obtained from milk produced by transgenic goats born from the goat impregnated with the embryo or progeny thereof. In order to increase an amount of the antibody-containing milk produced by the transgenic goats, hormones may be administered to the transgenic goats as appropriate (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

In the present invention, in addition to the aforementioned antibodies, an artificially modified, genetically recombinant antibody (e.g., a chimera antibody or a humanized antibody) may be employed. Such a modified antibody may be produced through a known method.

Specifically, a chimera antibody is prepared through the following procedure: the above-obtained DNA encoding the antibody V regions is ligated to the DNA encoding the human antibody C regions; the thus-ligated DNA is integrated into an expression vector; and the expression vector is introduced into a host for production of the chimera antibody. Through this known procedure, a chimera antibody useful for the present invention can be prepared.

A humanized antibody is also called a “reshaped human antibody” and is obtained by grafting a complementarity-determining regions (CDRs) of an antibody from a non-human mammal (e.g., mouse) into the complementarity-determining regions of a human antibody. Typical gene recombination techniques for preparing such a humanized antibody are known (see European Patent Application Laid-Open (EP) No. 125023 and WO 96/02576).

Specifically, a DNA sequence designed to ligate a CDRs of a mouse antibody to a framework regions (FRs) of a human antibody is synthesized through PCR employing, as primers, several oligonucleotides prepared to have portions overlapping terminal regions of both the CDRs and FRs (see the method described in WO 98/13388).

The framework regions of the human antibody ligated via the CDRs are selected in such a manner that the complementarity-determining regions form a proper antigen-binding site. If necessary, amino acid residues in the framework regions of the antibody variable regions may be substituted so that the complementarity-determining regions of a reshaped human antibody form a proper antigen-binding site (Sato, K., et al., Cancer Res. (1993) 53, 851-856).

The C regions employed in a chimera antibody or a humanized antibody may be those of a human antibody; for example, Cγ1, Cγ2, Cγ3, and Cγ4 in the H chain, and Cκ and Cλ in the L chain. The C regions of the human antibody may be modified so as to improve a stability of the antibody or to achieve stable production thereof.

The chimera antibody includes the variable regions of an antibody derived from a non-human mammal and the constant regions of a human antibody. Meanwhile, the humanized antibody includes the complementarity-determining regions of an antibody derived from a non-human mammal and the framework regions and C regions of a human antibody. The humanized antibody is useful as an active ingredient of a therapeutic agent, since it exhibits low antigenicity in a human body.

The anti-nicastrin antibody employed in the present invention is not limited to the whole antibody molecule. So long as the anti-nicastrin antibody binds to nicastrin protein, the antibody may be an antibody fragment, derivatives of the antibody (including a modified antibody, and an antibody bound to a compound exhibiting a desired pharmaceutical activity), a divalent antibody, or a monovalent antibody. The anti-nicastrin antibody is preferably an antibody which neutralizes human active γ-secretase.

Examples of the antibody fragment include Fab, F(ab′)₂, Fab/c having Fab having one Fv and complete Fc, and single-chain Fv (scFv) in which Fv fragments of the H or L chain are linked together with an appropriate linker. Specifically, an antibody is treated with an enzyme (e.g., papain or pepsin) to produce an antibody fragment. Alternatively, a gene encoding such an antibody fragment is constructed and introduced into an expression vector, followed by expression in an appropriate host cell (see, for example, Co, M. S., et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J., et. al., Methods in Enzymology (1989) 121, 663-669; and Bird, R. E., et al., TIBTECH (1991) 9, 132-137).

A single-chain Fv (scFv) is obtained by linking the H chain V region and L chain V region of an antibody. In the scFv fragment, the H chain V region and the L chain V region are linked by a linker (preferably, a peptide linker) (Huston, J. S., et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv fragment may be derived from any of the antibodies described herein. The peptide linker employed for linking the V regions is, for example, any single-stranded peptide including 12 to 19 amino acid residues.

DNA encoding the scFv fragment is obtained through PCR amplification employing, as a template, an entire sequence of the DNA encoding the H chain or H chain V region of the aforementioned antibody or the DNA encoding the L chain or L chain V region of the antibody, or a portion of the DNA sequence encoding an amino acid sequence of interest, in combination with a primer pair defining both ends of the DNA sequence, followed by amplification employing the DNA encoding a peptide linker region in combination with a primer pair which defines both ends of the DNA so that the respective ends are linked to the H and L chains.

Once the DNA encoding the scFv fragment is prepared, an expression vector containing the DNA and a host transformed with the expression vector can be obtained through a customary method, and the scFv fragment can be obtained through a customary method by use of the host.

Such an antibody fragment may be produced by a host after a gene for the fragment has been obtained and expressed in a manner similar to that described above. As used herein, the term “antibody” also encompasses such an antibody fragment.

Also, a modified anti-nicastrin antibody prepared through conjugation of a molecule (e.g., polyethylene glycol (PEG) or a sugar chain) to an anti-nicastrin antibody may be employed. Through such modification, a half-life of the anti-nicastrin antibody can be prolonged, and hydrolysis or elimination thereof can be reduced in blood. As used herein, the term “antibody” also encompasses such a modified antibody. Such a modified antibody may be prepared through chemical modification of the above-obtained antibody or a fragment thereof. Methods for modifying antibodies have already been established in the art.

Also, the antibody employed in the present invention may be a bispecific antibody. The bispecific antibody may have antigen-binding sites recognizing different epitopes of NCT molecule. A bispecific antibody may be prepared by binding HL pairs of two antibodies, or may be obtained from a bispecific-antibody-producing fused cell prepared through fusion of hybridomas producing different monoclonal antibodies. Alternatively, a bispecific antibody may be prepared through a genetic engineering technique.

The above-constructed gene for the antibody may be expressed through a known method, to thereby yield the antibody. In the case where a mammalian cell is employed, the antibody gene may be expressed by functionally binding a common useful promoter, the gene which is expressed, and a polyA signal downstream of a 3′-end thereof. Examples of the promoter/enhancer which may be employed include human cytomegalovirus immediate early promoter/enhancer.

Other promoters/enhancers which may be employed for antibody expression in the present invention include viral promoters/enhancers such as retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40); and promoters/enhancers derived from mammalian cells, such as human elongation factor 1α (HEF1α).

When SV40 promoter/enhancer is employed, gene expression can be readily carried out through a method of Mulligan, et al. (Nature (1979) 277, 108), whereas when HEF1α promoter/enhancer is employed, gene expression can be readily carried out through a method of Mizushima, et al. (Nucleic Acids Res. (1990) 18, 5322).

In the case where Escherichia coli are employed, the gene for the antibody can be expressed by functionally binding a common useful promoter, a signal sequence for secreting the antibody, and the antibody gene which is expressed. Examples of the promoter which may be employed include lacZ promoter and araB promoter. When lacZ promoter is employed, the gene can be expressed through a method of Ward, et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), whereas when araB promoter is employed, the gene can be expressed through a method of Better, et al. (Science (1988) 240, 1041-1043).

When the antibody is produced in a periplasm of Escherichia coli, a pe1B signal sequence (Lei, S. P., et al., J. Bacteriol. (1987) 169, 4379) may be employed as a signal sequence for secreting the antibody. The antibody produced in the periplasm is isolated and then employed by appropriately refolding a structure of the antibody.

Replication origins which may be employed include those derived from SV40, polyomavirus, adenovirus, bovine papilloma virus (BPV). In order to increase gene copy number in a host cell system, the expression vector employed may contain a selective marker such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

Any expression system such as a eukaryotic or prokaryotic system may be used for production of the antibody employed in the present invention. Examples of the eukaryotic cell include animal cells of, for example, established mammalian cell line, cells of insect cell line, filamentous fungal cells, and yeast cells; and examples of the prokaryotic cell include cells of a bacterium such as Escherichia coli.

Preferably, the antibody employed in the present invention is expressed in a mammalian cell such as CHO, COS, myeloma, BHK, Vero, or HeLa cell.

Subsequently, the above-transformed host cell is cultured in vitro or in vivo to produce a target antibody. Culturing of the host cell is carried out through a known method. For example, DMEM, MEM, RPMI 1640, or IMDM may be employed as a culture medium, and a serum supplement such as fetal calf serum (FCS) may be employed in combination.

The above-expressed or produced antibody can be isolated from cells or a host animal and purified to homogeneity. Isolation and purification of the antibody employed in the present invention may be carried out by means of an affinity column. Examples of columns employing protein A column include Hyper D, POROS, and Sepharose F.F. (products of Pharmacia). No particular limitation is imposed on the method for isolation/purification of the antibody, and the antibody may be isolated or purified through any method which is generally employed for isolation/separation of proteins. For example, the antibody may be isolated/purified by appropriately selecting or combining chromatography columns other than the aforementioned affinity columns, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

As described in the Examples hereinbelow, the above-obtained anti-nicastrin antibody recognizes nicastrin protein (i.e., a constituent molecule of human active γ-secretase), binds specifically to nicastrin protein, and exhibits an activity to neutralize γ-secretase. In addition, the anti-nicastrin antibody has an ability to inhibit proliferation of γ-secretase-dependent cancer cells. Therefore, the anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative is effective as a therapeutic drug for Alzheimer's disease and/or a cancer.

Conceivably, the cancer which can be treated by the present invention is a nicastrin-expressing cancer and/or a γ-secretase-dependent cancer.

Examples of such a cancer include lung cancer and T-cell acute lymphoblastic leukemia.

As used herein, “nicastrin-expressing cancer” refers to a cancer in which nicastrin protein is produced through expression of the nicastrin gene; and “γ-secretase-dependent cancer” refers to a cancer in which proliferation of cancer cells requires γ-secretase, and cancer cell proliferation is inhibited or cancer cells die through inhibition of γ-secretase activity.

The anti-nicastrin antibody derivative or a fragment thereof also encompasses a product prepared by conjugating a compound exhibiting a desired pharmaceutical activity to the anti-nicastrin antibody or a fragment thereof through a customary method. Such an anti-nicastrin antibody derivative may be employed in, for example, a missile therapy specifically targeting nicastrin. As used herein, “compound exhibiting a desired pharmaceutical activity” refers to a compound exhibiting, for example, a pharmaceutical activity to inhibit or promote a substance (e.g., an enzyme or a receptor) which directly or indirectly causes symptoms to progress.

Examples of compounds exhibiting a desired pharmaceutical activity for cancer treatment include a compound which causes damage to cancer cells, and a compound which provides or enhances cytotoxic activity (e.g., a radioisotope). The radioisotope employed may be any radioisotope known to those skilled in the art, but is preferably ¹³¹I, ^(99m)Tc, ¹¹¹In, or ⁹⁰Y.

Cancer treatment employing an antibody bound to a radioisotope-containing compound may be carried out through a method known to those skilled in the art. Specifically, firstly, a small amount of an antibody bound to a radioisotope-containing compound is administered to a patient, followed by whole-body scintigraphy. After determination that a degree of binding between the antibody and normal tissue cells is low but the degree of binding between the antibody and cancer cells is high, a large amount of the radioisotope-bound antibody is administered to the patient.

The therapeutic drug of the present invention may be prepared into a drug product by subjecting both the drug and a pharmaceutically acceptable carrier well known in the art to a drug preparation process such as mixing, dissolution, granulation, tableting, emulsification, encapsulation, or lyophilization.

For oral administration, the therapeutic drug of the present invention may be mixed with, for example, a pharmaceutically acceptable solvent, excipient, binder, stabilizer, or dispersant, and the mixture may be prepared into a dosage form such as tablet, pill, sugar-coated agent, soft capsule, hard capsule, solution, suspension, emulsion, gel, syrup, or slurry.

For parenteral administration, the therapeutic drug of the present invention may be mixed with, for example, a pharmaceutically acceptable solvent, excipient, binder, stabilizer, or dispersant, and the mixture may be prepared into a dosage form such as injection solution, suspension, emulsion, cream, ointment, inhalant, or suppository. For formulation of an injection, the therapeutic drug of the present invention may be dissolved in an aqueous solution, preferably, a physiologically compatible buffer (e.g., Hanks' solution, Ringer solution, or saline buffer). The composition may be in the form of suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the therapeutic drug may be produced in the form of powder, and, before use, the drug may be prepared into an aqueous solution or suspension with, for example, sterile water. For inhalation administration, the therapeutic drug of the present invention may be powdered and may be prepared into a powder mixture together with an appropriate base such as lactose or starch. For production of a suppository, the therapeutic drug of the present invention may be mixed with a conventional suppository base such as cocoa butter. The therapeutic drug of the present invention may be formulated into a sustained-release drug product by encapsulating the drug in, for example, a polymer matrix.

A dose of the therapeutic drug of the present invention or a number of doses thereof varies depending on a dosage form or administration route thereof, or the symptom, age, or body weight of a patient in need thereof. The therapeutic drug can be administered once to several times per day so that a daily dose of the drug is generally about 0.001 mg to about 1,000 mg per kg body weight, preferably about 0.01 mg to about 10 mg per kg body weight.

Generally, the therapeutic drug is administered through a parenteral route; for example, injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection), or transdermal, transmucosal, transnasal, or transpulmonary administration. However, no particular limitation is imposed on the administration route of the therapeutic drug, and the drug may be orally administered.

A screening method for selecting an antibody which inhibits γ-secretase activity.

As described in the Examples hereinbelow, an anti-nicastrin antibody has been found to inhibit reaction between nicastrin and a γ-secretase substrate (e.g., C99 or N99).

Therefore, the screening method of the present invention for selecting an antibody which inhibits γ-secretase activity is a promising method for searching a therapeutic drug for AD or cancer.

In the screening method of the present invention, nicastrin is reacted with a γ-secretase substrate (e.g., a polypeptide formed of the entirety or a portion of Notch receptor and/or APP (including the intramembrane sequence)) in the presence of a test antibody, and the reaction between nicastrin and the substrate is detected.

Specifically, nicastrin is reacted with a polypeptide formed of the entirety or a portion of the sequence of Notch or APP in the presence of a test antibody through addition thereof, and whether or not the added test antibody inhibits the reaction is determined through a known detection method.

Alternatively, a test antibody is exposed to cells expressing nicastrin and a polypeptide formed of the entirety or a portion of the sequence of Notch and/or APP, and whether or not a product is produced through the reaction between nicastrin and the polypeptide is determined through a known detection method.

Preferably, the latter screening method is carried out. The latter screening method requires a simpler screening process. In addition, when the latter screening method is carried out in combination with a known detection method, numerous test antibodies can be screened to determine whether or not they inhibit γ-secretase activity within a short period of time. Thus, a therapeutic drug for AD or cancer can be developed within a short period.

The γ-secretase substrate employed in the screening method may be a polypeptide formed of the entirety or a portion of Notch receptor (NM_(—)008714) (SEQ ID NO: 3) (including the intramembrane sequence) and/or a polypeptide formed of the entirety or a portion of APP protein (NM_(—)000484) (SEQ ID NO: 4) (including the intramembrane sequence). The polypeptide formed of the entirety or a portion of Notch receptor or APP protein may be prepared through expression of a gene having a sequence (5′-cacctcatgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtgc tgctg-3′) (SEQ ID NO: 6) and encoding a polypeptide including the intramembrane sequence of Notch receptor (NH₂-HLMYVAAAAFVLLFFVGCGVLL-COOH) (SEQ ID NO: 5), or a gene having a sequence (5′-ggtgcaatcattggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcacct tggtgatgctg-3′) (SEQ ID NO: 8) and encoding a polypeptide including the intramembrane sequence of APP protein (NH₂-GAIIGLMVGGVVIATVIVITLVML-COOH) (SEQ ID NO: 7). Particularly preferably, the polypeptide formed of the entirety or a portion of Notch receptor or APP protein is prepared through expression of a gene having a sequence (SEQ ID NO: 10) and encoding 99 amino acid residues (No. 1711 to No. 1809) of a protein of Notch receptor including the intramembrane sequence (NH₂-VKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASKK KRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) (SEQ ID NO: 9) (the 99 amino acid residues may be called “N99”), or a gene having a sequence (SEQ ID NO: 12) and encoding 99 amino acid residues (the C-terminus to No. 99) of a protein of APP including the intramembrane sequence (NH₂-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIH HGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN-COOH) (SEQ ID NO: 11) (the 99 amino acid residues may be called “C99”).

The polypeptide serving as a γ-secretase substrate, which is formed of the entirety or a portion of a protein, is obtained from the corresponding amino acid sequence or expressing a gene encoding the amino acid residues. Alternatively, the polypeptide is obtained from a natural product.

The polypeptide serving as a γ-secretase substrate, which is formed of the entirety or a portion of a protein, is preferably derived from human. However, the origin of the polypeptide is not limited to human, and the polypeptide may be derived from any non-human species such as dog, cat, mouse, hamster, or drosophila.

The amino acid sequence of nicastrin or a γ-secretase substrate or the sequence of the gene coding therefor may be provided, before expression thereof, with a tag sequence (e.g., V5 or FLAG sequence), which is selected in consideration of a detection method employed.

Whether or not a test antibody inhibits γ-secretase activity may be determined through a known technique such as co-immunoprecipitation (IP), western blotting, ELISA, reporter gene assay, a SPA beads method, a fluorescence polarization method, or a homogeneous time-resolved fluorescence method. These techniques may be employed singly or in combination as appropriate.

For example, co-immunoprecipitation (IP) and western blotting may be employed in combination. In this case, the amino acid sequence of nicastrin or a peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) or the sequence of the gene coding therefor is provided with a tag sequence (e.g., FLAG or V5 sequence) through a known method, and the protein or peptide is expressed in a host cell.

Nicastrin or the peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) is extracted from the host cell by a known extraction method including lysis of the cell membrane, followed by purification as appropriate.

The thus-extracted nicastrin is diluted with a culture medium and then mixed with a test antibody, and reaction is carried out at 4° C. for 8 to 12 hours. Thereafter, the Notch or APP peptide is added to the reaction mixture, followed by further mixing for three to four hours. A HEPES buffer containing 0.5% CHAPSO is employed as a buffer solution.

An antibody corresponding to the tag is added, and IP is carried out. Subsequently, a precipitated fraction is analyzed through a known western blot technique. The tag-corresponding antibody may be bound to a carrier (e.g., agarose beads) in advance.

In this case, when nicastrin and the peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) are precipitated in smaller amounts, the test antibody is determined to have higher percent inhibition of γ-secretase activity.

Alternatively, binding assay may be carried out by immobilizing one of nicastrin and the peptide on, for example, a carrier or an assay plate, and labeling the other with, for example, a radioisotope or a fluorescent substance. A test antibody detected may be provided with a tag (e.g., an antigen) or a label (e.g., a radioisotope).

Whether or not a test antibody inhibits γ-secretase activity may be determined through, for example, a method employing a GAL4-UAS system and ELISA or a reporter gene in combination. In this case, a construct (SC100G) is prepared by inserting GAL4 into C99 through a known method, and a reporter construct (UAS-luc) is prepared by inserting a UAS sequence into an upstream region of the luciferase gene serving as a reporter gene. These constructs are introduced into host cells through a known technique such as lipofection. Cells constitutively expressing nicastrin are selected by use of, for example, an antibiotic-resistant marker as appropriate.

The constitutively expressing cells are cultured at 37° C. for 24 hours, and then a test antibody is exposed to the cells, followed by expression of the transgene. Expression of the gene is induced by addition of 10 mM n-butylic acid. After culturing at 37° C. for 12 hours, the cells or the resultant supernatant is recovered.

In the case where the cells are employed, when the amount of luminescence generated by luciferase is reduced after lysis of the cells, the test antibody is determined to inhibit binding between nicastrin and a Notch receptor intramembrane peptide or an APP peptide, and to inhibit cleavage of the Notch receptor intramembrane peptide or the APP peptide; i.e., the test antibody is determined to have high percent inhibition of γ-secretase activity.

In the case where the supernatant is employed, an extracellularly released Aβ peptide fraction having an indicator applicable to ELISA is assayed through ELISA. When the degree of ELISA reaction is low, the test antibody is determined to inhibit binding between nicastrin and Notch receptor or an APP peptide, and to inhibit cleavage of the intramembrane sequence of a Notch receptor peptide or the APP peptide; i.e., the test antibody is determined to have high percent inhibition of γ-secretase activity.

Alkaline phosphatase or GFP may be employed in place of luciferase.

EXAMPLES

The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1 Culturing of Insect Cells

Insect cells (Spodoptera frugiperda, Sf9) were cultured at 27° C. by use of Grace's Insect Media Supplemented (Invitrogen) containing 10% fetal bovine serum (FBS, Sigma), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen). When mass culture was carried out, 0.001% pluronic F-68 (Invitrogen) was added to the aforementioned medium placed in a 1-L spinner flask.

Example 2 Preparation of Recombinant Virus

Human nicastrin cDNA cloned into pEF6-TOPO/V5-His (Invitrogen) (pEF6-NCT) (T. Tomita et al., FEBS Lett. 520 (2002) 117-121) was subcloned into pBlueBac4.5 (Invitrogen) so that the V5-His tag derived from the vector was provided on the C-terminal side, to thereby prepare a human-nicastrin-containing construct (pBlueBac4.5-NCT). Recombinant virus preparation was carried out according to a protocol attached Bac-N-Blue Transfection Kit (Invitrogen). Specifically, Sf9 cells were transfected with Bac-N-Blue DNA and the above-prepared plasmid (4 μg), followed by purification through a plaque assay (several times), to thereby prepare a recombinant virus containing only a target gene. After preparation of a high titer stock, a titer of the virus was determined through a plaque assay.

Example 3 Confirmation of Expression of Nicastrin on BV

Expression of nicastrin (i.e., a single-transmembrane protein) on BV was confirmed by use of the above-prepared recombinant virus. Sf9 cells were infected with the recombinant virus at a multiplicity of infection (MOI) of 5, and the cells and BV were recovered after 12 hours, 24 hours, 48 hours, or 72 hours initiation of infection, followed by confirmation of expression of nicastrin through immunoblotting by use of an anti-nicastrin N-terminal antibody (anti-NCT (N-19), SantaCruz) and an anti-His antibody. As a result, nicastrin was found to be sufficiently expressed in both a cell fraction and a BV fraction 48 hours after initiation of infection. This indicates that, similar to the case of SREBP-2 (Y. Urano, et al., Biochem. Biophys. Res. Commun. 308 (2003) 191-196), nicastrin is expressed on BV.

Example 4 Preparation of Anti-Nicastrin Antibody by Use of Budding Virus (BV)

Since a large amount of gp64, which is a virus-derived membrane protein and exhibits high antigenicity, is expressed on BV, when a mouse is infected with BV, an anti-gp64 antibody is strongly induced, and difficulty is encountered in yielding an antibody to a target antigen. Therefore, gp64 transgenic mice, which were prepared so as to exhibit resistance to gp64, were employed as mice for immunization.

Sf9 cells (5×10⁸ cells/500 mL) were infected with human-nicastrin-expressing recombinant virus (NCT-BV) at an MOI of 5, and cultured for 48 hours, followed by recovery of a culture supernatant. BV serving as an antigen was prepared from the culture supernatant through ultracentrifugation, and then gp64 transgenic mice were immunized five times with the antigen.

Screening of antisera and a resultant hybridoma culture supernatants was carried out through BV-ELISA by a customary method. There were added NCT-BV employed during immunization (serving as an antigen for immobilization), and SREBP+SCAP−BV prepared through coinfection of SREBP-2 and SREBP cleavage-activating protein (SCAP) (Y. Urano, et al., Biochem. Biophys. Res. Commun. 308 (2003) 191-196) or wild-BV containing no foreign gene (20 μg/mL in saline) (serving as a negative control) (50 μL/well). As a result, there were yielded many clones which do not respond to wild-type BV or SREBP/SCAP-expressing BV but show positive response to only nicastrin-expressing BV (FIG. 1). Similarly, there were yielded a plurality of clones (e.g., PPMX0401 and PPMX0410) which recognize nicastrin expressed on BV, as determined through immunoblotting employing BV (FIG. 2).

Example 5 Cell Culture

COS-7 cells (cells derived from simian kidney), HeLa cells (cells derived from human cervical cancer), A549 cells (cells derived from human lung cancer), or NKO cells (fibroblasts derived from nicastrin knockout mouse: T. Li, et al., J. Neurosci. 23 (2003) 3272-3277) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen) at 37° C. and 5% CO₂.

Example 6 Identification of Antibody by Use of BV and Forced Expression Product

The culture supernatants of positive clones selected through BV-ELISA were subjected to SDS-PAGE and immunoblotting by use of NCT-BV, Wild-BV, human wild-type nicastrin, and mutant forms of nicastrin in the presence of a 1×SDS-PAGE sample buffer. An anti-nicastrin N-terminal antibody (N-19) was employed as a positive control. In transient expression by use of animal cells, transfection into COS-7 cells was carried out by use of DEAE-dextran, and cells were recovered 48 hours after initiation of transfection. pEF6-NCT was employed for human wild-type nicastrin. Mutant nicastrin constructs (Δ312 and Δ694) were prepared from pEF6-NCT through long PCR (T. Tomita, et al., FEES Lett. 520 (2002) 117-121).

As a result, all the tested antibodies were found to recognize exogenously expressed human wild-type nicastrin. The anti-nicastrin N-terminal antibody (N-19) (i.e., a positive control) or an antibody to the C-terminal-added V5 tag recognized both wild-type nicastrin and mutant forms of nicastrin. In contrast, almost all the above-prepared antibodies (clones) (e.g., PPMX0401 and PPMX0410) did not recognize nicastrin Δ312 (FIG. 3). This suggests that the epitope site of each of the above-prepared antibodies is present in the extracellular domain of nicastrin.

Example 7 Preparation of Cells Constitutively Expressing Nicastrin

For the purpose of analysis of anti-nicastrin antibodies, NKO cells were transfected with pEF6-NCT by use of LipofectAmine (Invitrogen), and then NKO cells constitutively expressing human nicastrin (NKO/NCT cells) were selected in a medium containing 10 μg/mL blasticidin.

Example 8 Deglycosylation of Nicastrin

As has been known, nicastrin has, in the sequence thereof, 20 potential glycosylation sites and highly undergoes N-linked glycosylation (T. Tomita, et al., FEBS Lett. 520 (2002) 117-121; J. Y. Leem, et al., J. Biol. Chem. 277 (2002) 19236-19249; D. S. Yang, et al., J. Biol. Chem. 277 (2002) 28135-28142; and W. T. Kimberly, et al., J. Biol. Chem. 277 (2002) 35113-35117).

Nicastrin is classified, on the basis of the degree of glycosylation, into mature nicastrin (molecular weight: about 130 kDa) and immature nicastrin (molecular weight: about 110 kDa). Active γ-secretase complex contains only mature nicastrin. Among N-linked sugar chains, complex-type sugar chains are known to exhibit resistance to endoglycosidase H (Endo H) but to be cleaved by peptide: N-glycosidase F (PNGase). Therefore, through Endo H treatment, the molecular weight of complex-type glycosylated mature nicastrin is reduced to about 115 kDa, whereas the molecular weight of immature nicastrin having no complex-type sugar chain is reduced to about 80 kDa. In contrast, through PNGase F treatment, the molecular weights of both the mature nicastrin and immature nicastrin are reduced to about 80 kDa (D. S. Yang, et al., J. Biol. Chem. 277 (2002) 28135-28142, and W. T. Kimberly, et al., J. Biol. Chem. 277 (2002) 35113-35117).

In Example 8, a deglycosylation experiment was carried out for a purpose of epitopic analysis of anti-nicastrin antibodies.

Firstly, NKO/NCT cells were washed with PBS and then suspended in an RIPA buffer (50 mM Tris-HCl at pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl), followed by inversion mixing at 4° C. for eight hours for lysis. Nicastrin was immunoprecipitated (IP) from the resultant lysate fraction by use of an anti-nicastrin. C-terminal antibody (N1660, Sigma), and the thus-precipitated nicastrin fraction was employed for the following analysis.

For Endo H or PNGase treatment, 200 mM citrate-NaOH (pH 5.8), 0.1% SDS, and 1% 2-mercaptoethanol were added to the nicastrin fraction, and the mixture was boiled at 95° C. for five minutes. 500 mU/mL Endoglycosidase H (Roche Applied Sciences) or 200 U/mL PNGase F (Roche Applied Sciences) was added to the mixture, and reaction was carried out at 37° C. overnight. Finally, a 5× sample buffer (¼ amount of the reaction mixture) was added to the reaction mixture, and the resultant mixture was boiled at 95° C. for five minutes, whereby reaction was terminated.

For neuraminidase (sialidase) treatment, 50 mM Na-acetate (pH 5.2), 2 mM CaCl₂, and 0.5% 2-mercaptoethanol were added to the nicastrin fraction, and the mixture was boiled at 95° C. for five minutes. 500 mU/mL Neuraminidase (Roche Applied Sciences) was added to the mixture, and reaction was carried out at 37° C. overnight. Finally, a 5× sample buffer (¼ amount of the reaction mixture) was added to the reaction mixture, and the resultant mixture was boiled at 95° C. for five minutes, whereby reaction was terminated.

Samples prepared through treatment with the aforementioned deglycosylation enzymes were subjected to western blot analysis. As a result, PPMX0401, PPMX0408, and PPMX0410 were found to exhibit cross-reactivity to deglycosylated nicastrin (FIG. 4). In FIG. 4, “O” represents Endo H-resistant nicastrin; “black dot” represents completely deglycosylated nicastrin; and “Δ” represents neuraminidase-desialylated nicastrin.

These data (in particular, the fact that each of the above-prepared antibodies recognized nicastrin which had been completely deglycosylated by PNGase F) suggest that the antibody binds to nicastrin by recognizing a peptide chain of the protein rather than a sugar chain thereof.

Example 9 Immunoprecipitation (IP) of Endogenous Nicastrin by Use of Anti-Nicastrin Antibody

HeLa cells were suspended in a cell homogenization buffer (10% glycerol-containing HEPES buffer (10 mM HEPES pH 7.4, 150 mM NaCl complete inhibitor cocktail (Roche Applied Sciences))) and homogenized by means of a homogenizer, followed by centrifugation at 1,500×g for 10 minutes. Subsequently, the resultant supernatant was centrifuged at 100,000×g for one hour, and the precipitate was employed as a HeLa cell membrane fraction. The cell membrane fraction was lysed in a 1% CHAPSO-containing HEPES buffer, to thereby yield a HeLa cell membrane lysate fraction. After IP of nicastrin from the lysate fraction by use of each of the above-prepared anti-nicastrin monoclonal antibodies, western blot analysis was carried out by use of various antibodies.

As a result, the above-prepared antibodies were found to be classified into two groups; i.e., antibodies which allow IP of only immature nicastrin (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) (first group); and antibodies which allow IP of both immature nicastrin and mature nicastrin (PPMX0406, PPMX0408, and PPMX0410) (second group) (FIG. 5). In the case of the antibodies of the second group (PPMX0406, PPMX0408, and PPMX0410), presenilin, PEN-2, and APH-1aL, which are components of the γ-secretase complex, were coprecipitated, whereas in the case of the antibodies of the first group (PPMX0401, PPMX0402, PPMX0407, and PPMX0409), only APH-1aL was precipitated in a small amount. As has been reported, immature nicastrin binds to APH-1 and forms a sub-complex before it forms a γ-secretase complex (M. LaVoie, et al., J. Biol. Chem. 278 (2003) 37213-37222). Therefore, conceivably, each of the antibodies of the first group (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) binds specifically to immature nicastrin contained in a nicastrin-APH-1 sub-complex.

As has also been reported, the structure of the extracellular domain of nicastrin changes with formation of the γ-secretase complex (K. Shirotani, et al., J. Biol. Chem. 278 (2003) 16474-16477). When the HeLa cell 1% CHAPSO lysate was treated with trypsin, the extracellular domain of nicastrin exhibited resistance to trypsin. Therefore, conceivably, the extracellular domain of nicastrin maintains its structure in the γ-secretase complex in the presence of 1% CHAPSO (FIG. 6). Thus, the data of the IP experiment suggest that the epitope site of each antibody of the first group is masked through structural change of nicastrin, whereas the epitope site of each antibody of the second group may be exposed even after structural change of nicastrin.

Example 10 Immunostaining of Cultured Cells by Use of Anti-Nicastrin Monoclonal Antibody

Biochemical studies have reported that active γ-secretase containing mature nicastrin is localized to lipid rafts (Urano Y., Hayashi I., Isoo N., et al.: Association of active γ-secretase complex with lipid rafts. J. Lipid Res. 2005, 46: 904). In this Example, cultured cells were immunostained by use of the above-prepared antibodies, to thereby examine intracellular localization of nicastrin recognized by the antibodies. Intracellular localization of nicastrin was examined by use of HeLa cells and NKO cells. Cells were bonded, at an appropriate cell density, to a cover glass which had been coated with poly-D-lysine in advance, and the cells were washed with PBS and then fixed with PBS containing 4% paraformaldehyde. PBS containing 3% BSA was employed for blocking, and, in the case of permeation, Triton X-100 (final concentration: 0.1%) was further added. Each of the antibodies was diluted to an appropriate concentration with a blocking solution, and the thus-diluted antibody was reacted with the cells (at room temperature for three hours, or at 4° C. overnight). An anti-mouse or anti-rabbit immunoglobulin antibody bound to Alexa 488 or 546 was employed as a secondary antibody.

As a result, in HeLa cells, a granular structure and the cell membrane were stained in the presence of PPMX0408, whereas such a structure was not stained in the presence of PPMX0401 (FIG. 7). In NKO cells, a granular structure was not stained in the presence of PPMX0408. However, when wild-type nicastrin was introduced into NKO cells, there was obtained a stained image similar to that obtained in the case of HeLa cells (FIG. 8). These data indicate that the granular structure stained in the presence of PPMX0408 is derived from nicastrin.

Subsequently, in order to examine intracellular localization of the granular structure, co-staining was carried out by use of PPMX0408 and antibodies to various marker proteins. As a result, localization of the granular structure did not correspond to that of calnexin and giantin, which are marker proteins for endoplasmic reticulum and Golgi body, respectively (FIG. 9). In contrast, the results of staining in the presence of cholera toxin subunit B (CTB), which is used for staining of GM1 ganglioside present in lipid rafts, corresponded well to those of staining of the granular structure in the presence of PPMX0408. The results of staining in the presence of PPMX0401 did not correspond to those of staining in the presence of CTB (FIG. 10). Correspondence of localization similar to that described above was observed even under non-permeating conditions (i.e., no treatment with Triton X-100 during blocking) (FIG. 10). These data suggest that PPMX0408 recognizes mature nicastrin which is localized to lipid rafts (including cell membrane).

Example 11 Neutralization of Human Active γ-Secretase Activity by Use of Anti-Nicastrin Monoclonal Antibody

Since PPMX0408 or PPMX0410 binds to mature nicastrin contained in active γ-secretase under the conditions where the γ-secretase complex is maintained, these antibodies are considered to affect γ-secretase activity. Therefore, a microsomal fraction of HeLa cells was lysed with 1% CHAPSO; each of the antibodies was added to an in vitro γ-secretase assay system employing an artificial substrate; and γ-secretase activity was determined on the basis of accumulation of de novo synthesized Aβ (Takasugi N., Tomita T., Hayashi I., Tsuruoka M., Niimura M., Takahashi Y., Thinakaran G., Iwatsubo T.: The role of presenilin cofactors in the γ-secretase complex. Nature 2003, 422: 438; and Takahashi Y., Hayashi I., Tominari Y., et al.: Sulindac sulfide is a non-competitive γ-secretase inhibitor that preferentially reduces Aβ 42 generation. J. Biol. Chem. 2003, 278: 18664).

When PPMX0401 was added (final concentration: 10 μg/mL), γ-secretase activity was maintained at almost the same level as in the case where PBS was added. In contrast, when PPMX0408 or PPMX0410 was added (final concentration: 10 μg/mL), γ-secretase activity was inhibited by about 20%, as compared with the case where PBS was added (FIG. 11). This suggest that the antibodies which bind to mature nicastrin exhibit γ-secretase inhibitory activity.

Example 12 Effect of Anti-Nicastrin Monoclonal Antibody on Viability of Cancer Cell Lines

Firstly, in order to identify a cancer cell line exhibiting Notch-signaling-dependent survival, the effect of a γ-secretase inhibitor DAPT (H F. Dovey, et al., J. Neurochem. 76 (2001) 173-181) on survival of HeLa cells or A549 cells was evaluated through the MTT method. HeLa cells or A549 cells (5×10³ cells) were inoculated onto a 96-well multiplate and treated with DAPT (final concentration: 100 μM) for 72 hours. After the 72-hour treatment, MTT diluted with PBS was added to the plate so that the final MTT concentration was 500 μg/mL, followed by culturing at 37° C. for three to four hours. Thereafter, stop solution (10% SDS, 0.01 M HCl) was added to the plate for termination of reaction, and the plate was allowed to stand still at 37° C. overnight, followed by dissolution of produced formazan. The formazan solution was uniformly mixed through pipetting, and absorbance was measured at 550 nm, to thereby calculate cell viability. As a result, the viability of DAPT-treated A549 cells was significantly lower than that of DAPT-untreated A549 cells. In contrast, no significant difference was observed in viability between DAPT-treated HeLa cells and DAPT-untreated HeLa cells (FIG. 12). Subsequently, in order to confirm that this reduction in cell viability was attributed to inhibition of γ-secretase activity, endogenous nicastrin of A549 cells was knocked down through treatment with nicastrin-corresponding short interference RNA (siRNA), and change in cell viability was determined. As a result, in the case of treatment with nicastrin siRNA, cell viability was reduced by about 20%, as compared with the case of treatment with siRNA having a random sequence (scramble) (FIG. 13). Under the nicastrin siRNA treatment conditions, expression of endogenous nicastrin was completely inhibited (FIG. 14). These data suggest that, unlike the case of HeLa cells, survival of A549 cells requires γ-secretase activity.

Subsequently, the effect of the above-prepared antibodies on survival of A549 cells was examined. Each of the antibodies was added to A549 cells so that the final antibody concentration was 10 μg/mL, and, 96 hours after addition of the antibody, cell viability was determined through the MTT method. As a result, the viability of PPMX0410-treated A549 cells was significantly lower than that of antibody-untreated A549 cells or PPMX0401-treated A549 cells (FIG. 15). These data suggest that an antibody exhibiting γ-secretase inhibitory activity has an ability to inhibit proliferation of cancer cells exhibiting γ-secretase-dependent survival.

Example 13 Effect of Anti-Nicastrin Monoclonal Antibody on Proliferation of Leukemia Cell Lines

As has been reported, proliferation of cells of the following cell lines: TALL-1, ALL-SIL, and DND-41—which are isolated and established from patients with T-cell acute lymphoblastic leukemia (T-ALL)—requires Notch signaling (Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P. t., Silverman, L. B., Sanchez-Irizarry, C., Blacklow, S. C., Look, A. T. and Aster, J. C. (2004), Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269-271). As has also been reported, in TALL-1 cells, somatic mutation is not found in the Notch1 gene, but in ALL-SIL cells or DND-41 cells, missense mutation occurs in the region (HDN) involved in interaction between an extracellular domain of Notch1 and TMIC (transmembrane-intracellular domain of Notch), and deletion (by mutation) occurs in the PEST region involved in degradation of NICD (Notch intracellular domain) (FIG. 16). Conceivably, mutation of the HDN region causes ligand-independent heterodimeric dissociation, shedding, and cleavage by γ-secretase, and deletion in the PEST region increases the stability of NICD, which induces abnormal activation of Notch signaling, thereby causing T-ALL.

Firstly, there was examined the effect of treatment of TALL-1, ALL-SIL, or DND-41 cells with a γ-secretase inhibitor on metabolism of Notch1. Through western blot analysis by use of an antibody mN1A to the intracellular ankyrin repeat domain of Notch1 (Chemicon, Cat #MAB5352), a band considered to be attributed to Notch1 TMIC was observed in the cases of all these types of cells. In the case of ALL-SIL cells or DND-41 cells, a band considered to be attributed to NEXT (Notch extracellular truncation) was observed at a position slightly below the TMIC band, and also a somewhat unclear band considered to be attributed to NICD was observed at a position below the NEXT band (FIG. 17). In the case of ALL-SIL cells or DND-41 cells, constitutive expression of NICD was determined by an antibody Val1744 (Cell Signaling, Cat #2421) specific to the cleaved N-terminal of NICD, but in the case of TALL-1 cells, expression of NICD was not observed. Subsequently, a γ-secretase inhibitor YO (concentration: 10, 100, or 1,000 nM) was added to the culture supernatant of each type of cells, and the cells were recovered 48 hours after addition of YO, followed by western blot analysis of the resultant lysate. As a result, in the case of YO treatment of ALL-SIL cells or DND-41 cells, NICD was found to disappear, and TMIC and NEXT were found to be accumulated (FIG. 17). These data suggest that Notch signaling is constitutively activated in at least both ALL-SIL cells and DND-41 cells.

Subsequently, the effect of YO treatment on proliferation of these cells was examined. Cells were inoculated onto a 96-well plate (5×10³ cells/well) and cultured at 37° C. overnight. Then, a γ-secretase inhibitor YO was added to the plate, followed by culturing for seven days. Thereafter, percent cell proliferation was determined by use of Alamar Blue (Serotec). Alamar Blue was added to the culture liquid in an amount of 1/10 that of the culture liquid, followed by culturing at 37° C. for four hours. Subsequently, the resultant culture supernatant was recovered. Fluorescence in the culture supernatant was measured by means of a plate reader (excitation wavelength: 530 nm, fluorescence wavelength: 590 nm), and percent cell proliferation was calculated by use of the following formula.

${{cell}\mspace{14mu} {proliferation}\mspace{14mu} (\%)} = {\frac{A_{590}}{{PC}_{590}} \times 100}$

In the above formula, “A590” represents the absorbance of a sample at 590 nm, and “PC590” represents the absorbance of a positive control group (treated with PBS or DMSO) at 590 nm. As a result, proliferation of TALL-1 cells or DND-41 cells was found to be inhibited through YO treatment. Specifically, through treatment with 10 nM YO, proliferation of TALL-1 cells or DND-41 cells was inhibited by about 60% or about 50%, respectively, and, through treatment with 1,000 nM YO, proliferation of TALL-1 cells or DND-41 cells was inhibited by about 80% (FIG. 18). Unexpectedly, virtually no inhibition of cell proliferation was observed in ALL-SIL cells, in which NICD was found to disappear through YO treatment (as determined by western blot analysis). These data indicate that TALL-1 cells or DND-41 cells exhibit γ-secretase activity-dependent proliferation.

The above-obtained data suggest that, among the examined T-ALL-derived cells, at least DND-41 cells exhibit Notch signaling/γ-secretase activity-dependent proliferation. Therefore, the effect of PPMX0410 (i.e., an anti-nicastrin antibody) on proliferation of DND-41 cells was examined. PPMX0410 or a mouse IgG fraction (concentration: 0.1, 1, 10, or 100 μg/mL) was added to a DND-41 cell culture supernatant, followed by culturing for seven days. Thereafter, percent cell proliferation was determined by use of Alamar Blue. As a result, percent cell proliferation tended to slightly increase in an IgG-fraction-concentration-dependent manner, but tended to lower through addition of PPMX0410. Specifically, proliferation of DND-41 cells was inhibited by about 60% through addition of 100 μg/mL PPMX0410 (FIG. 19). These data indicate that PPMX0410 inhibits Notch signaling/γ-secretase activity-dependent proliferation of T-ALL cells.

On the basis of these results, PPMX0410 was deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6th, Tsukuba Center, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan, Postal Code 305-8566) (deposition date: Apr. 21, 2006, accession number: FERM-AP 20895).

Example 14 Effect of Anti-Nicastrin Monoclonal Antibody in Inhibiting Binding Between Nicastrin and Substrate

As has been reported, nicastrin may function as a substrate receptor in the γ-secretase complex (Shah S., Lee S F., Tabuchi K., Hao Y H., Yu C., LaPlant Q., Ball H., Dann C E 3rd, Sudhof T., Yu G.: Nicastrin functions as a γ-secretase-substrate receptor. Cell 2005, 122: 435).

Therefore, there was examined a possibility that PPMX0410 exhibits γ-secretase inhibitory activity by inhibiting interaction between γ-secretase and a substrate therefor.

Firstly, there was expressed, in Sf9 cells, nicastrin (having a V5-His tag sequence added at the carboxyl terminus) or N100-FLAG (100 amino acid residues (No. 1711 to No. 1809) of Notch receptor including the intramembrane sequence (NH₂-MVKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASK KKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) and having a FLAG-His tag (DYKDDDDKGSHHHHHH) added at the carboxyl terminus) (SEQ ID NOs: 13 and 14), Lee S F., Shah S., Li H., Yu C., Han W., Yu G.: Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-β precursor protein and Notch. J. Biol. Chem. 2002 277: 45013). Subsequently, a cell membrane fraction was prepared through the method described above in Example 9.

The resultant cell fraction was lysed in a HEPES buffer containing 1% CHAPSO, to thereby yield a nicastrin fraction or an N100 fraction.

The nicastrin fraction was mixed with PPMX0401 or PPMX0410 diluted to an appropriate concentration with PBS, and reaction was carried out at 4° C. overnight. Thereafter, the N100 fraction was added to the reaction mixture, followed by inversion mixing for three hours. A 1% CHAPSO-containing HEPES buffer was employed during mixing of the nicastrin fraction with the antibody, and a 0.5% CHAPSO-containing HEPES buffer was employed after addition of the N100 fraction.

Nicastrin and N100-FLAG were coprecipitated from the resultant nicastrin-N100 fraction mixture by use of anti-V5-antibody-bound V5-agarose beads (SIGMA) or anti-FLAG-antibody-bound M2-agarose beads (SIGMA), and the precipitated fraction was subjected to western blot analysis by use of an anti-V5 antibody (FIG. 20).

In the case where a nicastrin fraction which had been denatured with 0.1% SDS in advance was employed, the amount of nicastrin precipitated by the M2-agarose beads (i.e., nicastrin bound to N100-FLAG) was reduced, as compared with the case where a native nicastrin fraction was employed (comparison between lanes “D” and “N” in FIG. 20).

Thus, this experiment system was considered to be applicable to detection of structure-dependent binding of nicastrin to N100.

Under the aforementioned conditions, PPMX0401 or PPMX0410 was added, and the amount of nicastrin precipitated by the M2-agarose beads was measured, followed by comparison of the resultant measurement data. In the case where PPMX0410 was added at a concentration of 10 or 100 μg/mL, the amount of nicastrin precipitated was found to be considerably reduced (FIG. 21). When the measurement data were normalized with the amount of nicastrin precipitated by the V5-agarose beads, PPMX0410 (at the aforementioned concentrations) was found to inhibit binding between nicastrin and N100-FLAG by about 60% (FIG. 21).

These data suggest that PPMX0410 inhibits γ-secretase activity by inhibiting binding between nicastrin and a substrate for the enzyme. Thus, these data suggest that an antibody exhibiting potent γ-secretase inhibitory activity can be selected on the basis of inhibition of binding between nicastrin and a substrate for the enzyme.

Example 15 Inhibition of γ-Secretase Activity by Anti-Nicastrin Antibody in Living Cells

An experiment was carried out by means of a GAL4-UAS system employing reporter cells, in order to determine whether or not PPMX0410—which inhibits γ-secretase activity in an in vitro reaction system—also inhibits cleavage mediated by γ-secretase activity in living cells.

C99 is a fragment produced through cleavage of APP (amyloid precursor protein) by BACE (β-site APP cleaving enzyme) and serves as a direct substrate for γ-secretase.

A preproenkephalin-derived signal peptide was inserted into C99, and GAL4 (i.e., a yeast-derived transcription factor) was inserted immediately downstream of the transmembrance domain of C99, to thereby prepare a construct (SC100G). The construct (SC100G) was subcloned into pcDNA3.1/Hygro vector (Invitrogen).

GAL4/VP16 was bound to NΔE (including N99), in which deletion occurs in the extracellular domain of Notch receptor and which serves as a direct substrate for γ-secretase in a ligand-independent manner, to thereby prepare a construct (NΔEGV, Taniguchi Y., Karlstrom H., Lundkvist J., Mizutani T., Otaka A., Vestling M., Bernstein A., Donoviel D., Lendahl U., Honjo T.: Notch receptor cleavage depends on but is not directly executed by presenilins. Proc. Natl. Acad. Sci. U.S.A 2002, 99: 4014). The construct (NΔEGV) was subcloned into pcDNA3.1 vector (pcDNA3.1-NΔEGV).

A UAS sequence was inserted upstream of luciferase in pGL3(R2.2) vector (Promega), to thereby prepare a construct (UAS-luc), and the construct was employed as a reporter construct. eGFP was subcloned into pcDNA3 (Invitrogen), and the thus-prepared pcDNA3-eGFP was employed as a control vector for monitoring the number of cells. HEK293 cells were transfected with pcDNA3.1-SC100G and UAS-luc, or transfected with pcDNA3.1-NΔEGV, UAS-luc, and pcDNA3-eGFP by use of Lipofectamine 2000 (Invitrogen). Cells constitutively expressing nicastrin (HEK/SC100G cells or HEK/NΔEGV cells) were selected by use of an antibiotic-resistant marker (Hygromycin (Wako Pure Chemical Industries, Ltd.) or G418 (CALBIOCHEM), respectively).

HEK/SC100G cells or HEK/NΔEGV cells were inoculated into a 48-well multiplate (2.5×10⁴ cells). After culturing at 37° C. for 24 hours, PBS or PPMX0410 diluted to an appropriate concentration with PBS was added to the plate. Cells treated with DMSO or DAPT (final concentration: 10 μM) (i.e., γ-secretase-activity-inhibiting control) were also provided. After culturing at 37° C. for 36 hours, n-butyric acid (final concentration: 10 mM) was added for induction of transgene expression. After culturing for 12 hours, cells and a culture supernatant were recovered, and the amount of Aβ was determined through a reporter assay or ELISA.

The recovered cells were lysed in a lysis buffer (Promega), and the resultant lysate was subjected to the reporter assay. PicaGene (Toyo Ink Mfg. Co., Ltd.) was employed as a luminescent substrate. The amount of luciferase luminescence was normalized by the concentration of protein (in the case of HEK/SC100G cells), or by the amount of eGFP luminescence (in the case of HEK/NΔEGV cells), to thereby yield relative light unit (RLU). The amount of Aβ secreted in the culture supernatant of HEK/SC100G cells was determined through ELISA, and the thus-determined Aβ amount was normalized by the concentration of protein similar to the case of normalization of the amount of luciferase luminescence.

As a result, PPMX0410 was found to inhibit, in a concentration-dependent manner, reporter activity (FIG. 22A) and Aβ secretion (FIG. 22B) in HEK/SC100G cells, and reporter activity (FIG. 22C) in HEK/NΔEGV cells. Under the aforementioned conditions, DAPT (i.e., a γ-secretase inhibitor) was found to inhibit reporter activity in both HEK/SC100G cells and HEK/NΔEGV cells.

These data indicate that PPMX0410 also inhibits γ-secretase activity in living cells and inhibits intramembrane protein cleavage in APP or Notch receptor.

The method described in Example 14 can be employed for high throughput screening. Therefore, the method is considered applicable to selection of an antibody exhibiting potent γ-secretase inhibitory activity. 

1-9. (canceled)
 10. A method for treatment of Alzheimer's disease, comprising administering an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative to a subject in need thereof. 11-13. (canceled)
 14. The method of claim 10, comprising administering an anti-nicastrin antibody.
 15. The method of claim 10, comprising administering an antigen binding fragment of the anti-nicastrin antibody. 